BOORSE, GC; CRESPI, EJ; DAUTZENBERG, FM; DENVER, RJ; Univ of Michigan; Univ of Michigan; Johnson & Johnson, R&D; Univ of Michigan: Molecular cloning and analysis of Xenopus laevis urocortin 1 and urocortin 3

Vertebrates have several corticotropin-releasing factor (CRF)-like peptides that bind to and activate two G protein-coupled receptors (CRF₁ and CRF₂) to modulate endocrine, autonomic, and behavioral responses to stress. Mammals have four CRF-like peptides: CRF, urocortin 1 (UCN1), UCN2 and UCN3. In teleost fishes, CRF, urotensin I (a fish ortholog of mammalian UCN1) and UCN3 have been identified. In amphibians, CRFs have been isolated from five anuran species, but sauvagine is the only urotensin I/UCN-like peptide known. We isolated cDNAs for two urocortins, UCN1 and UCN3, from the South African clawed frog Xenopus laevis. Also, by analyzing genomic databases we discovered novel CRF-like peptides in pufferfish and chicken. Molecular phylogenetic analysis of all known vertebrate CRF-like peptides and those identified by us supports the existence of four paralogous lineages in tetrapods that likely arose before the radiation of the teleost fishes. RT-PCR analysis showed that UCN1 and UCN3 mRNAs are expressed in brain, pituitary, heart, and kidney; UCN1 but not UCN3 mRNA is expressed in skin. Based on their deduced amino acid sequences, we synthesized the Xenopus urocortins and studied their ligand-binder pharmacology and physiology. Xenopus CRF₁ and CRF-binding protein (CRF-BP) had higher affinity for CRF than for UCN1; whereas, CRF₂ bound UCN1 with similar affinity as CRF. Similar to mammals, Xenopus UCN3 is a selective ligand for CRF₂ and is not bound by the CRF-BP. ICV injections of both UCN1 and UCN3 inhibited food intake, thus supporting a role for the CRF₂ in the appetite-suppressive effects of CRF-like peptides in amphibians as has been shown in mammals. (Supported by NSF grant IBN 9974672 to RJD and NSF predoctoral fellowship to GCB).